CD28 is the only costimulatory receptor that is effective in assisting the T-cell antigen receptor (TCR) in proliferative expansion, IL-2 secretion and survival. Since this suggests a unique mechanism of costimulation, it is relevant that CD28 induces clustering of detergent-resistant, cholesterol-enriched lipid rafts at the TCR-associated supramolecular activation clusters. While it has been shown that raft signaling facilitates protein tyrosine kinase (PTK) activation, it is not clear how rafts assist in the engagement of specific downstream signaling cascades. In this application, we will use our unique system of generating large numbers of primary human CD4+ T-cells to test the hypothesis that CD28 is responsible for activation of the NF-kappaB and Jun kinase (JNK) cascades through its affects on lipid rafts and the associated cytoskeleton. We propose that senescent T-cells fail to properly assemble macromolecular signaling complexes because of a breakdown of this mechanism, thereby contributing to deficient T-cell activation in elderly people. In order to accomplish our long-term goal of understanding the mechanism of CD28 costimulation and functional decline during aging, we will test the hypothesis that CD28 has unique effects on the recruitment and activation of NF-kappaB and JNK cascade components to TCR-associated lipid rafts and the cortical cytoskeleton. In Aim 1 we will test the hypothesis that activation of the NF-kappaB and JNK cascades by CD28 costimulation depends on the association of cascade components with the ordered lipid phase (rafts) in primary human CD4+ T-cells. We will determine how altered assembly of the NF-kappaB cascade components in association with TCR-associated rafts may contribute to signaling decline during aging. In Aim 2, we will determine how the assembly of the NF-kappaB and JNK signaling components in lipid rafts leads to the activation of those cascades during CD28 costimulation. This will accomplished by large scale raft preparation from primary human T-cells and analysis of released signaling complexes by Western blotting, protein mass spectrometry and antibody arrays. On the functional level, we will determine how mutation of the anchor/adaptor proteins, IKKgamma and LAD, affect the activation of the NF-kappaB and JNK cascades, respectively. In Aim 3, we will identify the intracellular CD28 domain that, through communication with the cytoskeleton and the flotillins, directs rafts to the surface membrane and the TCR. This includes a study of flotillins as key raft structural proteins that communicate with the cytoskeleton and plays a role in raft expression on the cell surface. We will compare T-cells from young and elderly human subjects to determine how altered cytoskeletal assembly contributes to signaling decline by the TCR.